In recent years, in the field of biopharmaceuticals represented by, for example, antibody drugs, technologies for expression of target substances such as proteins are rapidly developed. Accordingly, improvement of productivity in the purification processes, based on chromatography etc., has been desired.
In order to carry out purification of biopolymers such as proteins with high efficiency, carriers for chromatography require high pressure resistance which enables use of the carrier at high flow rates, and require high binding capacity for biopolymers in the case where ligands are contained. Conventionally, as carriers for chromatography, for example, inorganic materials such as silica; synthetic polymers such as poly(meth)acrylic esters and poly(meth)acrylamides; natural polymers such as polysaccharides have been used.
Silica particles and synthetic polymer particles have high strength, and are therefore preferable for use at high flow rates. However, those particles usually exhibit low binding capacity to biopolymers when ligands are introduced, and the particles also cannot exhibit preferable resistance to alkaline solutions which are used for washing the carriers. Furthermore, since synthetic polymer particles are generally hydrophobic to a certain extent, these particles sometimes exhibit non-specific interactions, and consequently cause contamination of impurities.
As for natural polymers, polysaccharides such as agarose and cellulose are usually used. For example, agarose can be dissolved in water at a high temperature, and when the solution is subsequently cooled to a certain temperature (gelling point), a porous gel is formed therefrom. Therefore, agarose gel particles can be produced by dispersing an aqueous phase containing dissolved agarose in an organic solvent to form liquid droplets, and then cooling the dispersion to a temperature lower than or equal to the gelling point.
Furthermore, regarding cellulose, some methods for dissolving and regenerating cellulose to produce porous gel particles, for example, a method comprising dissolving cellulose in an aqueous solution of calcium thiocyanate (Patent Document 1), a method comprising dissolving cellulose in a lithium chloride solution of dimethylacetamide or N-methylpyrrolidone (Patent Document 2), a method comprising dissolving cellulose in an ionic liquid (Non-Patent Document 1), have been reported.
Since such polysaccharide gel particles contain a large number of hydroxyl groups in the molecule, their surfaces are highly hydrophilic. Therefore, those gel particles basically do not cause non-specific interactions with biological materials, and as a result, target molecules may be obtained in high degree of purity. Furthermore, in general, when compared with carriers prepared from silica or a synthetic polymer, the polysaccharide gel particles exhibit an excellent binding capacity for biological molecules, and excellent resistance to alkaline solutions which are generally used for washing filler for chromatography.
However, when polysaccharide gel particles are used as a carrier of filler for chromatography, the polysaccharide gel particles generally have lower mechanical strength as compared with silica particles or synthetic polymer particles. As a result, if the polysaccharide gel particles are used at a high flow rate, the particles are deformed within the column, causing loss of flow channels, and consequently, compaction which causes a limitless increase in the back pressure of the column will easily occur. Thus, the polysaccharide gel particles have a problem of poor resistance to flow rate.
As described above, various materials for chromatographic carriers have their unique defects.
In recent years, a number of means for overcoming those defects have been proposed. For example, a means for improvement of alkali resistance by coating the surface of silica with a hydrophobic siloxane compound (Patent Document 7), a method comprising a step of forming particles from a monomer ester having a bulky, highly hydrophobic aliphatic substituent and a step of modifying surface of the particle with a bis- or polyepoxide compound under alkaline conditions to provide hydrophilicity thereto (Patent Document 8), have been reported.
On the other hand, in regard to natural polymers, the most general method of increasing the mechanical strength and resistance to flow rate of polysaccharide gel particles involves crosslinking of the polysaccharide gel particles after particle formation. Such crosslinking is formed between hydroxyl groups inside the polysaccharide gel particles, and is generally performed by using a crosslinking agent such as epichlorohydrin. Regarding the formation of crosslinking, various methods for improvement have been hitherto reported, and for example, it has been reported that the crosslinking agarose gel particles with a bifunctional or polyfunctional crosslinking agent having a chain length of 6 to 12 atoms and then crosslinking the product with a bifunctional crosslinking agent having a chain length of 2 to 5 atoms to improve mechanical strength of crosslinked agarose gel particles (Patent Document 3); and that introducing dextran to the surface of crosslinked agarose particles, and further allowing the product to react with vinyl sulfonate for the introduction of a cation exchange group to obtain cation-exchange particles having an enhanced binding capacity (Patent Document 9).
Furthermore, it has been reported that crosslinking cellulose gel particles by using a crosslinking agent in which hydroxyl groups are present between functional groups to obtain a porous carrier having appropriate compressive stress (Patent Document 4); that sequentially introducing a crosslinking agent and an alkali solution to a suspension liquid containing cellulose gel particles to obtain cellulose gel particles having superior resistance to flow rate as compared with the case of introducing the entire amount of a crosslinking agent all at once (Patent Document 5); and the like.
Also, as a method of more efficiently crosslinking the interior of particles, there is a method of introducing in advance functional groups to a polysaccharide used as a raw material, subsequently forming particles, subsequently activating the functional groups, and thereby crosslinking the polysaccharide chains. According to this method, as compared with the method of performing crosslinking after the formation of particles, crosslinking can be implemented even inside a fine structure where it is difficult for a crosslinking agent to penetrate therein, so that it is therefore expected to obtain higher mechanical strength. For example, it is described in Patent Document 6 that when agarose is allowed to react with the epoxy group of allyl glycidyl ether to form gel particles, subsequently the allyl group of allyl glycidyl ether is activated and crosslinked with polysaccharide, the mechanical strength of crosslinked agarose gel particles can be improved. However, the technology requires multiple stages of reaction processes ranging from modification of agarose to crosslinking, and thus the operation is complicated. Furthermore, since the reaction between agarose and the epoxy group of allyl glycidyl ether is carried out in water, there is a defect that inactivation of the epoxy group may be caused by water in the reaction system, and the reaction efficiency is decreased.
As such, there are a number of technologies that may be utilized in the production of filler for chromatography which use polymer particles as carriers; however, a technology for producing a chromatographic carrier which achieves a good balance between high binding capacity for materials to be purified, and high pressure resistant performance that enables processing at a high flow rate, has not yet been sufficiently established. Along with the growth of the biopharmaceutical market in recent years, there is a rapidly increasing demand for high speed processing in the purification processes for target substances. In this technical field, there is a strong demand for a method for producing a filler for chromatography which exhibits both higher resistance to flow rate and higher binding capacity.